The importance of maintaining a balance between inflammatory responses necessary for pathogen clearance and their timely resolution to prevent tissue damage is clearly exemplified by patients with severe COVID-19, who develop life-threatening hyper-inflammation characterized by high levels of complement activation. Complement itself has been shown to drive both the differentiation of interferon-γ (IFNγ)-producing T helper 1 (TH1) cells as well as the eventual shutdown of their pro-inflammatory features through IL-10 expression. This study by Chauss et al. links the complement-mediated retraction of TH1 cell responses to vitamin D receptor (VDR) signalling, which provides a possible mechanistic explanation for the epidemiological associations between vitamin D deficiency and adverse outcomes in COVID-19 and potentially other infectious diseases.
Single-cell RNA sequencing of CD4+ T cells from the blood and bronchoalveolar lavage fluid (BALF) of eight patients with COVID-19 showed that they have a prominent pro-inflammatory TH1 cell signature, enriched for IFNγ and complement pathway genes, but with lower levels of expression of IL10 than CD4+ T cells from healthy controls. To better understand the pro-inflammatory nature of these COVID-19-associated TH1 cells, the authors looked in more detail at the previously described effects of complement on contraction of TH1 cell responses. Transcriptomic analysis of CD4+ T cells activated with anti-CD3 and anti-CD46 (the receptor for complement component C3b) showed upregulation of both VDR and CYP27B1, which encodes the enzyme catalysing the final activation step of vitamin D. This suggests that complement stimulates T cells to both activate and respond to vitamin D. T cells from CD46-deficient individuals or T cells treated with an inhibitor that blocks intracellular generation of C3b did not upregulate VDR or CYP27B1. In response to inactive vitamin D, T cells stimulated with anti-CD3 and anti-CD46 repressed IFNγ expression and upregulated IL-10 expression, suggesting that complement-induced VDR signalling promotes shutdown of TH1 cell responses.
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Further transcriptomic and proteomic analysis showed that vitamin D promotes the production of IL-10 by inducing expression of IL-6, IL-6 receptor (IL-6R) and the transcription factor STAT3, leading to STAT3 phosphorylation and activation downstream of IL-6R. Vitamin D also induced genome-wide changes in histone acetylation and recruitment of transcription factors, including at STAT3, IL10 and BACH2 loci. A significant proportion of vitamin D-driven transcription was shown to be BACH2 dependent, including IL6R transcription. Indeed, vitamin D-treated CD4+ T cells from a BACH2 haploinsufficient individual did not upregulate the expected vitamin D-dependent gene set, and the IL-6–STAT3–IL-10 signalling axis was disrupted.
complement stimulates T cells to both activate and respond to vitamin D
Given the observed association of COVID-19 severity with vitamin D deficiency, the authors looked at the expression of vitamin D-regulated genes in the BALF CD4+ T cells of patients with COVID-19. On a per-cell basis, they observed a reciprocal relationship between the expression of TH1 cell-associated genes and vitamin D-modulated genes. The results suggest that in patients with severe COVID-19, who have a strongly pro-inflammatory TH1 cell phenotype, the transcriptional response to vitamin D is impaired, leading to a failure to switch to IL-10 production and inflammatory resolution. This could be a result of vitamin D insufficiency or some other dysregulation of complement-induced autoregulatory VDR signalling; in either case, clinical trials may help shed light on whether there is clinical benefit to using vitamin D as an adjunct therapy for COVID-19. Furthermore, IL-6 is generally thought to be a pro-inflammatory cytokine involved in cytokine storm responses, but the results also suggest that vitamin D as adjunct therapy for COVID-19 might divert IL-6 to pro-resolution functions and thus provide an alternative to blocking IL-6R signalling. The researchers did not test vitamin D as an adjunct treatment for COVID-19 and caution the results should not be taken as a clinical recommendation.